The Adipose Tissue Core will provide a wide variety of cost-effective services for the study of human and mouse adipose tissue. Services will include techniques of cell separation and sorting, histology, molecular biology and protein chemistry. Specifically, freshly isolated human and mouse fat samples will be subjected to collagenase digestion (under conditions least likely to alter gene expression) to separate primary adipocytes from the stromal vascular fraction. Adipose tissue macrophages will subsequently be separated from the stromal vascular fraction with antibodycoated magnetic beads. Gene expression will be analyzed in whole adipose tissue and its cellular components using quantitative 'real-time'reverse-transcriptase polymerase chain reaction (rt-PCR, Roche LightCycler). Quantification of RNA copy number will be derived from plasmid standard curves for every gene of interest. Analysis of gene expression will be complemented by analysis of protein expression of key adipocyte secretory products by Western blotting. Additionally, Western blotting will be used to study the activation of various components of the insulin signaling pathway in adipose tissue sampled under a variety of in vivo conditions. Quantitative assessment of macrophage infiltration into adipose tissue will be performed by two alternative methods: quantitative rt-PCR (using specific macrophage markers) and Fluorescence Activated Cell Sorting (FACS) analysis. Since many of the above techniques require freshly obtained adipose tissue, the concentration of these techniques in one Core should favor efficiency and minimize handling. The following are several additional, highly specialized techniques offered by the Core. Adipocyte size and number will be quantified following osmium fixation of adipose tissue samples. Serial biopsies of rat adipose tissue will be performed in vivo by Dr. Vasselli. Lipoprotein lipase activity will be assayed in frozen adipose tissue samples by Dr. Johnson. Recombinant adipokine production will be provided by Dr. Lawrence Shapiro.